Cell lines expressing a mch receptor

ABSTRACT

The present invention features neuroblastoma and skin cell carcinoma cell lines functionally expressing MCHR1 and the use of such cells to measure MCHR1 activity. Functional expression is preferably achieved in a neuroblastoma or skin cell carcinoma using a recombinant gene expressing MCHR1. The presence of a recombinant MCHR1 gene increases the level of MCHR1 expression facilitating the production and detection of MCHR1 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplication U.S. Ser. No. 60/244,700, filed Oct. 31, 2000, herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The references cited herein are not admitted to be prior art tothe claimed invention.

[0003] Neuropeptides present in the hypothalamus play a major role inmediating the control of body weight. (Flier, et al., 1998. Cell, 92,437440.) Melanin-concentrating hormone (MCH) is a cyclic 19-amino acidneuropeptide synthesized as part of a larger pre-prohormone precursor inthe hypothalamus which also encodes neuropeptides NEI and NGE. (Nahon,et al., 1990. Mol. Endocrinol. 4, 632-637.) MCH was first identified insalmon pituitary, and in fish MCH affects melanin aggregation thusaffecting skin pigmentation. In trout and in eels MCH has also beenshown to be involved-in stress induced or CRF-stimulated ACTH release.(Kawauchi, et al., 1983. Nature 305, 321-323.)

[0004] In humans two genes encoding MCH have been identified that areexpressed in the brain. (Breton, et al., 1993. Mol. Brain Res. 18,297-310.) In mammals MCH has been localized primarily to neuronal cellbodies of the hypothalamus which are implicated in the control of foodintake, including perikarya of the lateral hypothalamus and zonainertia. (Knigge, et al., 1996. Peptides 17, 1063-1073.)

[0005] Pharmacological and genetic evidence suggest that the primarymode of MCH action is to promote feeding (orexigenic). MCH mRNA is upregulated in fasted mice and rats, in the ob/ob mouse and in mice withtargeted disruption in the gene for neuropeptide Y (NPY). (Qu, et al.,1996. Nature 380, 243-247, and Erickson, et al., 1996. Nature 381,415-418.) Injection of MCH centrally (ICV) stimulates food intake andMCH antagonizes the hypophagic effects seen with α melanocytestimulating hormone (αMSH). (Qu, et al., 1996. Nature 380, 243-247.) MCHdeficient mice are lean, hypophagic and have increased metabolic rate.(Shimada, et al., 1998. Nature 396, 670-673.)

[0006] MCH action is not limited to modulation of food intake as effectson the hypothalamic-pituitary-axis have been reported. (Nahon, 1994.Critical Rev. in Neurobiol. 8, 221-262.) MCH may be involved in the bodyresponse to stress as MCH can modulate the stress-induced release of CRFfrom the hypothalamus and ACTH from the pituitary. In addition, MCHneuronal systems may be involved in reproductive or maternal function.

[0007] Several references describe a receptor that is indicated to bindMCH (human MCHR1). (Chambers, et al., 1999. Nature 400, 261-265; Saito,et al., 1999. Nature 400, 265-269; Bäichner, et al., 1999. FEBS Letters457, 522-524; Shimomura, et al., 1999. Biochemical and BiophysicalResearch Communications 261, 622-626; and Lembo, et al., 1999. Nat. CellBiol. 1, 267-27 1.)

SUMMARY OF THE INVENTION

[0008] The present invention features neuroblastoma and skin cellcarcinoma cell lines functionally expressing MCHR1 and the use of suchcells to measure MCHR1 activity. Functional expression is preferablyachieved in a neuroblastoma or skin cell carcinoma using a recombinantgene expressing MCHR1. The presence of a recombinant MCHR1 geneincreases the level of MCHR1 expression facilitating the production anddetection of MCHR1 activity.

[0009] Thus, a first aspect of the present invention describes aneuroblastoma or skin cell carcinoma comprising a recombinant MCHR1 genethat expresses functional MCHR1. Functional MCHR1 produces a detectablesignal upon MCH stimulation. The recombinant MCHR1 gene may be part of agenome or may be present outside of the genome.

[0010] An MCHR1 gene contains nucleic acid encoding for MCHR1 andregulatory elements needed for functional expression. Examples ofregulatory elements useful for functional expression include a promoter,a terminator, a ribosome binding site, and a polyadenylation region. Thenucleic acid encoding for MCHR1 can be contiguous or may contain one ormore introns.

[0011] A recombinant MCHR1 gene encodes for MCHR1 and contains one ormore regions not naturally associated with each other. Examples ofrecombinant MCHR1 genes include those containing a human nucleic acidsequence encoding for MCHR1 present with a regulatory sequence notnaturally associated with the encoding nucleic acid; and thosecontaining a non-naturally occurring encoding region. A non-naturallyencoding region contains one or more combinations of nucleotides notpresent in the naturally occurring encoding nucleic acid. Recombinantgenes can be produced with, or without, intron(s).

[0012] Another aspect of the present invention describes a neuroblastomaor skin cell carcinoma having increased MCHR1 expression produced by aprocess comprising the step of coupling endogenous nucleic acid encodingfor MCHR1 to an exogenous promoter. The process results in theproduction of a recombinant MCHR1 gene having the same chromosomallocation as the native MCHR1 gene.

[0013] Another aspect of the present invention describes a method ofmeasuring the ability of a compound to affect MCHR1 activity. The methodcomprises the steps of: (a) providing a compound to a neuroblastoma orskin cell carcinoma expressing MCHR1; and (b) measuring MCHR1 activity.

[0014] Other features and advantages of the present invention areapparent from the additional descriptions provided herein including thedifferent examples. The provided examples illustrate differentcomponents and methodology useful in practicing the present invention.The examples do not limit the claimed invention. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Human neuroblastoma and skin cell carcinoma cell lines able toproduce MCHR1 transcripts and to provide a suitable environment formeasuring MCHR1 activity are identified herein. Human cell linesexpressing MCHR1 provide a human cellular environment that naturallyexpresses MCHR1.

[0016] Cells expressing functional MCHR1 provide a system for screeningfor compounds active at MCHR1, measuring the effect of a compound atMCHR1, and measuring the effect of MCHR1 activity. MCHR1 expression in aneuroblastoma or skin cell carcinoma is preferably increased using arecombinant MCHR1 gene that either makes use of endogenous nucleic acidencoding for MCHR1 or provides exogenous nucleic acid encoding MCHR1.

[0017] Compounds modulating MCHR1 activity have a variety of differentuses including utility as a tool to further study MCHR1 activity and asan agent to achieve a beneficial effect in a patient. Beneficial effectsof modulating MCHR1 activity include one or more of the following:weight loss, weight gain, cancer treatment (e.g., colon or breast), painreduction, diabetes treatment, stress reduction and sexual dysfunctiontreatment.

[0018] Modulating MCHR1 activity includes evoking a response at thereceptor and altering a response evoked by a MCHR1 agonist orantagonist. Generally, MCH receptor antagonists and allostericmodulators negatively affecting activity may be used to achieve weightloss, treat cancer (e.g., colon or breast), reduce pain, reduce stressor treat sexual dysfunction; and MCH receptor agonists and allostericmodulators positively affecting activity may be used to produce a weightgain.

[0019] A patient is a mammal, preferably a human. Reference to patientdoes not necessarily indicate the presence of a disease or disorder. Theterm patient includes subjects treated prophylactically and subjectsafflicted with a disease or disorder.

[0020] Preferably, MCHR1 activity is modulated to treat diabetes, toobtain a weight loss, or to obtain a weight gain. Diabetes mellitus canbe treated by, for example, one or both of the following: enhancingglucose tolerance and decreasing insulin resistance.

[0021] Excessive weight is a contributing factor to different diseasesincluding hypertension, diabetes, dyslipidemias, cardiovascular disease,gall stones, osteoarthritis and certain forms of cancers. Bringing abouta weight loss can be used, for example, to reduce the likelihood of suchdiseases and as part of a treatment for such diseases. Weight reductioncan be achieved by, for example, one or more of the following: reducingappetite, increasing metabolic rate, reducing fat intake or reducingcarbohydrate craving.

[0022] Increasing weight is particularly useful for a patient having adisease or disorder, or under going a treatment, accompanied by weightloss. Examples of diseases or disorders accompanied by weight lossinclude anorexia, AIDS, wasting, cachexia, and frail elderly. Examplesof treatments accompanied by weight loss include chemotherapy andradiation therapy.

MCHR1

[0023] MCHR1 employed in the present invention includes naturallyoccurring human MCHR1 having the amino acid sequence provided by SEQ.ID. NO. 1 and variants thereof. Variants of MCHR1 have a substantiallyidentical amino acid sequence as SEQ. ID. NO. 1 and have MCH receptoractivity. Examples of SEQ. ID. NO. 1 variants include naturallyoccurring allelic variants and artificially produced mutants.

[0024] SEQ. ID. NO. 1 and the naturally occurring encoding nucleic acidsequence were initially identified as the somatostatin-like receptor“SLC-1.” (Lakaye, et al., 1998. Biochimica et Biophysica ACTA1401:216-220.) Subsequently, SLC-1 was shown to be MCHR1. cDNA andgenomic sequences encoding for MCHR1 are provided by SEQ. ID. NOs. 2 and3.

[0025] In general, SEQ. ID. NO. 1 variants have a sequence identity ofat least about 90%, preferably, at least about 95%, with SEQ. ID. NO. 1;and/or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modificationsfrom SEQ. ID. NO. 1. Amino acid modifications are additions, deletions,and substitutions.

[0026] Sequence similarity for polypeptides can be determined by BLAST.(Altschul, et al., 1997. Nucleic Acids Res. 25, 3389-3402, herebyincorporated by reference herein.) In one embodiment, sequencesimilarity is determined using tBLASTn search program with the followingparameters: MATRIX:BLOSUM62, PER RESIDUE GAP COST: 11, and Lambda ratio:1.

[0027] Artificial variants of MCHR1 can be produced in a cell byintroducing nucleic acid encoding for the variant. Nucleic acidsequences encoding for variants can be obtained by altering the nucleicacid sequence encoding for SEQ. ID. NO. 1. The translation of aparticular codon into a particular amino acid is well known in the art(see, e.g., Lewin, GENES IV, p. 119, Oxford University Press, 1990):

[0028] A=Ala=Alanine: codons GCA, GCC, GCG, GCU

[0029] C=Cys=Cysteine: codons UGC, UGU

[0030] D=Asp=Aspartic acid: codons GAC, GAU

[0031] E=Glu=Glutamic acid: codons GAA, GAG

[0032] F=Phe=Phenylalanine: codons WUC, UUU

[0033] G=Gly=Glycine: codons GGA, GGC, GGG, GGU

[0034] H=His=Histidine: codons CAC, CAU

[0035] I=Ile=Isoleucine: codons AUA, AUC, AUU

[0036] K=Lys=Lysine: codons AAA, AAG

[0037] L=Leu=Leucine: codons WUA, WUG, CUA, CUC, CUG, CUU

[0038] M=Met=Methionine: codon AUG

[0039] N=Asn=Asparagine: codons AAC, AAU

[0040] P=Pro=Proline: codons CCA, CCC, CCG, CCU

[0041] Q=Gln=Glutamine: codons CAA, CAG

[0042] R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

[0043] S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

[0044] T=Thr=Threonine: codons ACA, ACC, ACG, ACU

[0045] V=Val=Valine: codons GUA, GUC, GUG, GUU

[0046] W=Trp=Tryptophan: codon UGG

[0047] Y=Tyr=Tyrosine: codons UAC, UAU

[0048] Changes to SEQ. ID. NO. 1 to produce functional variants may beempirically determined. Techniques for measuring MCH receptor activityare well known in the art.

[0049] One method of producing functional variants of SEQ. ID. NO. 1expected to retain some MCH receptor activity takes into accountdifferences in amino acid R groups. An R group effects differentproperties of an amino acid such as physical size, charge, andhydrophobicity. Amino acids can be divided into different groups asfollows: neutral and hydrophobic (alanine, valine, leucine, isoleucine,proline, tryptophan, phenylalanine, and methionine); neutral and polar(glycine, serine, threonine, tyrosine, cysteine, asparagine, andglutamine); basic (lysine, arginine, and histidine); and acidic(aspartic acid and glutamic acid).

[0050] Generally, in substituting different amino acids it is preferableto exchange amino acids having similar properties. Substitutingdifferent amino acids within a particular group, such as substitutingvaline for leucine, arginine for lysine, and asparagine for glutamineare good candidates for not causing a change in polypeptide functioning.

[0051] Changes outside of different amino acid groups can also be made.Preferably, such changes are made taking into account the position ofthe amino acid to be substituted in the polypeptide. For example,arginine can substitute more freely for nonpolor amino acids in theinterior of a polypeptide then glutamate because of its long aliphaticside chain. (See, Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987-1998, Supplement 33 Appendix IC.)

Recombinant MCHR1 Gene

[0052] A recombinant MCHR1 gene can be used to increase the level ofMCHR1 expression in a neuroblastoma or skin cell carcinoma therebyfacilitating the production and detection of MCHR1 activity. Methods forproducing a recombinant MCHR1 gene include those altering the endogenousMCHR1 gene and those introducing an MCHR1 gene or coding region into ahost cell.

[0053] Alterations of an endogenous MCHR1 gene producing a recombinantgene include the use of regulatory elements such as a promoter orenhancer not naturally associated with the MCHR1 coding region; andusing a non-naturally encoding region containing one or morecombinations of nucleotides not present in the naturally occurringencoding nucleic acid. Examples of exogenous promoters include the humancytomegalovirus promoter (“CMV”), α-MHC promoter, PrP (prion promoter),potent neuronal promoter and Thy-1 promoter.

[0054] Non-naturally occurring encoding regions can be produced based onthe degeneracy of the genetic code. If desired, the nucleic acidencoding for MCHR1 can be altered based on the genetic code to adjustcodon frequency.

[0055] Techniques that can be used for creating a recombinantchromosomal gene are well known in the art. (See Ausubel, CurrentProtocols in Molecular Biology, John Wiley, 1987-1998). Exogenousnucleic acid can be targeted to the MCHR1 gene using homologousrecombination targeting sequences. Homologous recombination targetingsequences for insertion into the MCHR1 gene include coding andnon-coding regions.

[0056] An exogenous promoter, such as the CMV promoter, can befunctionally coupled to MCHR1 nucleic acid using standard techniques.For example, the GOTO MCHR1 genomic sequence can be cloned into aplasmid vector. The MCHR1 promoter region (2-3 kb) upstream of codingsequence can be replaced by a CMV promoter cassette containing aloxP-neomycin-loxP gene. The resulting promoter-exchange vector wouldhave sufficient MCHR1 genomic sequences flanking the CMV promotercassette for homologous recombination. The vector can then beelectroporated into the GOTO cell. Neomycin resistant clones can beselected and verified by genomic Southern analysis for successfulpromoter exchange. The neomycin gene can be removed by loxp mediatedrecombination to reduce its possible interference with promoteractivity.

[0057] Introducing an MCHR1 gene or coding region into a host can beachieved by inserting a MCHR1 coding region or gene into the host genomeor through the use of an independently replicating vector. Techniquesfor inserting nucleic acid into the host genome include those targetingand selecting for insertion in a particular region and those involvingrandom insertion.

Functional Assays

[0058] Techniques for measuring MCHR1 activity include detecting achange in the intracellular conformation of MCHR1, measuring G-proteinactivity, and measuring the level of intracellular messengers. Assaysmeasuring different G-protein activities, such as Gi, Gs, and Gq can becarried out using techniques that are well known in the art. MCHR1activity is preferably assayed for by measuring either Gi or Gqactivity.

[0059] Gi and Gs activity can be measured using techniques such as amelonaphore assay, assaying cAMP production, assaying inhibition of cAMPaccumulation, intracellular acidification, and assaying 35S-GTP binding.cAMP can be measured using different techniques such as aradioimmunoassay and indirectly by cAMP responsive gene reporterproteins.

[0060] Gq and Gi activity can be measured using techniques such as thosedetecting intracellular Ca²⁺ and intracellular acidification. Examplesof techniques well known in the art that can be employed to measure Ca²⁺include the use of dyes such as Fura-2 and the use ofCa²⁺-bioluminescent sensitive reporter proteins such as aequorin.(Button, et al., 1993. Cell Calcium 14, 663-671, and Feighner, et al.,1999. Science 284, 2184-2188, both of which are hereby incorporated byreference herein.)

[0061] Functional assays can be performed using individual compounds orpreparations containing different compounds. A preparation containingdifferent compounds where one or more compounds affect MCHR1 activitycan be divided into smaller groups of compounds to identify thecompound(s) affecting MCHR1 activity. In an embodiment of the presentinvention a test preparation containing at least 10 compounds is used ina functional assay.

MCHR1 Expressing Cell Lines

[0062] Human cell lines able to express MCHR1 transcripts includeneuroblastoma and skin cell carcinoma. The ability of differentneuroblastoma cell lines and a squamous cell carcinoma is illustrated inthe Examples provided below. The squamous cell carcinoma provides anexample of a skin cell carcinoma. In different embodiments of thepresent invention concerning a skin cell carcinoma, the skin cellcarcinoma is a squamous cell carcinoma or a kertinocyte cell carcinoma.

[0063] The ability of different neuroblastoma cells lines and a skincell carcinoma to express MCHR1 transcripts points to these types ofcell lines as containing members able to express MCHR1. Additionalneuroblastoma and skin cell carcinoma cell lines able to express MCHR1transcripts can be identified using routine experimentation, forexample, by measuring the ability of neuroblastoma and skin cellcarcinoma cell lines present in depositories such as American TypeCulture Collection (Virginia, U.S.) and Health Science ResearchResources Bank (Osaka, Japan) to express MCHR1 transcripts.

Modulating MCHR1 Activity

[0064] Using the present application as a guide compounds able tomodulate MCHR1 activity can be obtained and used as a research tool tofurther explore the affects of MCHR1 activation or as a therapeutic toachieve a beneficial effect in a patient. Beneficial effects can beobtained, for example, by using a compound active at MCHR1 to achieveone or more of the following: weight loss, weight gain, treat cancer(e.g., colon or breast), reduce pain, treat diabetes, reduce stress orteat sexual dysfunction.

[0065] Altering weight is particularly useful for gaining weight in anunder weight patient or losing weight in an over weight patient. Inaddition, for example, farm animals can be treated to gain weight. Underweight patients include those having a body weight about 10% or less,20% or less, or 30% or less, than the lower end of a “normal” weightrange or Body Mass Index (“BMI”). Over weight patients include thosehaving a body weight about 10% or more, 20% or more, 30% or more, or 50%or more, than the upper end of a “normal” weight range or BMI. “Normal”weight ranges are well known in the art and take into account factorssuch as a patient age, height, and body type.

[0066] BMI measures your height/weight ratio. It is determined bycalculating weight in kilograms divided by the square of height inmeters. The BMI “normal” range is 19-22.

[0067] MCHR1 modulating compounds can be provided in kit. Such a kittypically contains an active compound in dosage form for administration.A dosage form contains a sufficient amount of active compound such thata beneficial effect can be obtained when administered to a patientduring regular intervals, such as 1 to 6 times a day, during the courseof 1 or more days. Preferably, a kit contains instructions indicatingthe use of the dosage form to achieve a beneficial effect and the amountof dosage form to be taken over a specified time period.

Dosing For Therapeutic Applications

[0068] Guidelines for pharmaceutical administration in general areprovided in, for example, Reminington's Pharmaceutical Sciences 18^(th)Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics2^(nd) Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, bothof which are hereby incorporated by reference herein.

[0069] MCHR1 active compounds having appropriate functional groups canbe prepared as acid or base salts. Pharmaceutically acceptable salts (inthe form of water- or oil-soluble or dispersible products) includeconventional non-toxic salts or the quaternary ammonium salts that areformed, e.g., from inorganic or organic acids or bases. Examples of suchsalts include acid addition salts such as acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate;and base salts such as ammonium salts, alkali metal salts such as sodiumand potassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine.

[0070] MCHR1 active compounds can be administered using different routesincluding oral, nasal, by injection, and transmucosally. Activeingredients to be administered orally as a suspension can be preparedaccording to techniques well known in the art of pharmaceuticalformulation and may contain microcrystalline cellulose for impartingbulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents. As immediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants.

[0071] When administered by nasal aerosol or inhalation, compositionscan be prepared according to techniques well known in the art ofpharmaceutical formulation. Examples of formulation components includesolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents.

[0072] The compounds may also be administered in intravenous (both bolusand infusion), intraperitoneal, subcutaneous, topical with or withoutocclusion, or intramuscular form. When administered by injection, theinjectable solutions or suspensions may be formulated using suitablenon-toxic, parenterally-acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

[0073] When rectally administered in the form of suppositories,compositions may be prepared by mixing the drug with a suitablenon-irritating excipient, such as cocoa butter, synthetic glycerideesters or polyethylene glycols, which are solid at ordinarytemperatures, but liquidify and/or dissolve in the rectal cavity torelease the drug.

[0074] Suitable dosing regimens for the therapeutic applications can beselected taking into account factors well known in the art includingage, weight, sex and medical condition of the patient; the severity ofthe condition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular compound employed.

[0075] Optimal precision in achieving concentrations of drug within therange that yields efficacy without toxicity requires a regimen based onthe kinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug. The daily dose for a patient is expected to be between 0.01 and1,000 mg per adult patient per day.

EXAMPLES

[0076] Examples are provided below to further illustrate differentfeatures of the present invention. The examples also illustrate usefulmethodology for practicing the invention. These examples do not limitthe claimed invention.

Example 1 MCHR1 Expression in Different Cell Lines

[0077] RT-PCR experiments were performed to determine whether differenthuman cell lines expressed mRNA for the MCHR1. Purified poly (A)+mRNAwas isolated from cultured cells using an oligo-dT kit (Poly (A) Pure,Ambion, Austin, Tex.). RT-PCR using ˜1 μg of isolated mRNA was performedusing Superscript II reverse transcriptase (Life Technologies,Gaithersberg, Md.) essentially following the manufacturer'sinstructions. PCR cycling conditions were: 94° C. for 1 minute (onecycle), 94° C. for 30 seconds then 72° C. for 4 minutes (four cycles);94° C. for 30 seconds then 70° C. for 4 minutes (four cycles); 94° C.for 30 seconds then 68° C. for 4 minutes (25 cycles); and 68° C. for 10minutes (one cycle). PCR primers were chosen based on the human MCHR1DNA sequence to flank the mRNA splice junction on either side of thesingle intron between exon 1 and exon 2. Forward sense primers had thefollowing sequences: SEQ. ID. NO. 4: ATGGACCTGGAAGCCTCGCTGCTG, SEQ. ID.NO. 5: GCCAGCAACACCTCTGATGGC, and SEQ. ID. NO. 6:GGCCCCGATAACCTCACTTCGGC. Reverse (anti sense) primers had the followingsequences: SEQ. ID. NO. 7: GAGGAGATCTACTACCGAGAGG, SEQ. ID. NO. 8:GCCCATGAGCTGGTGGATCATG and SEQ. ID. NO. 9: GTGGACAGTGGCCAGGTAGAGGTC.

[0078] Amplified products were electrophoresed on an agarose gel, andSouthern blotted with a human MCHR1 radiolabeled probe. The probe wasrandom prime labeled with ³²P. Post-hybridization washing stringency wasat 65° C., 1×SSC, after which the filters were dried and exposed toX-ray film for 3 hours at −70° C. The results are shown in Table 1.TABLE 1 Cell Line ATCC No. Type RT-PCR Results HGT-1 Gastric carcinoma −H4 Neuroglioma − TE671 Medulloblastoma − SK-N-BE2 CRL-2271Neuroblastoma + T-98G Glioblastoma − U-87MG Glioblastoma − CCF-DTTGAstrocytoma − MC-IXC Neuroblastoma − SMS-KAN Neuroblastoma − SMS-MSNNeuroblastoma − CHP-212 CRL-2273 Neuroblastoma + CHP-243 CRL-2272Neuroblastoma + IMR32 Neuroblastoma − GT1-7 Hypothalamus (SV- − 40immortalized) SH-SY5Y CRL-2266 Neuroblastoma + SCC-25 CRL-1628 Squamouscell + carcinoma GOTO 1 1 Adrenal + Neuroblastoma

[0079] Neuroblastoma cells lines GOTO, CHP-212, CHP-243, SK-N-BE(2), andSH-SY5Y were found to produce mRNA encoding for human MCHR1. Inaddition, the squamous cell carcinoma cell line SCC-25 was found toproduce mRNA encoding for human MCHR1.

Example 2 MCH Receptor Activity in Neurobastoma Cells Lines

[0080] To assess whether neuroblastoma cells provide an environment forfunctional MCHR1, MCH receptor activity was measured using GOTO cellsemploying an aequorin bioluminescence assay. The aequorinbioluminescence assay can be used to measure the activity of Gprotein-coupled receptors that couple through the Ga protein subunitfamily consisting of Gq, G11, and Gi leading to the activation ofphospholipase C, mobilization of intracellular calcium and activation ofprotein kinase C. Measurement of functional MCHR1 expression in GOTOcells transiently expressing aequorin was performed using a LuminoskanRT luminometer (Labsystems Inc., Gaithersburg, Md.) controlled by customsoftware written for a Macintosh PowerPC 6100.

[0081] GOTO cells (1.2×10⁷ cells plated 18 hours before transfection ina T75 flask) were transfected with human MCHR1 plasmid DNA and aequorincDNA using the Lipofectamine 2000 procedure (Life Technologies,Gaithersburg, Md.). Human MCHR1 plasmid DNA contained the open readingframe cDNA (SEQ. ID. NO. 2) encoding the human MCHR1 receptor insertedin the mammalian expression vector pcDNA-3 (Invitrogen, Carlsbad,Calif.). An aequorin expressing plasmid contained the cDNA for aequorin(Button, et al., 1993. Cell Calcium 14, 663-671), inserted in pcDNA-3.Following approximately 40 hours of expression the apo-aequorin in thecells was charged for 1 hour with coelenterazine (10 μM) under reducingconditions (300 μM reduced glutathione) in ECB buffer (140 mM NaCl, 20mM KCl, 20 mM HEPES-NaOH [pH=7.4], 5 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂,0.1 mg/ml bovine serum albumin). The cells were harvested, washed oncein ECB medium and resuspended to 500,000 cells/ml or 1,000,000cells/ml.

[0082] 100 μl of MCH or, for control responses, lysophosphatidic acidwere injected into a cell suspension (corresponding to 5×10⁴ cells or100,000 cells). Lysophosphatidic acid triggers native edg receptorscoupled to PLC activation present on GOTO cells. The integrated lightemission was recorded over 30 seconds, in 0.5 second units. 20 μL oflysis buffer (0.1% final Triton X-100 concentration) was then injectedand the integrated light emission recorded over 10 seconds, in 0.5second units. The “fractional response” values for each well werecalculated by taking the ratio of the integrated response to the initialchallenge to the total integrated luminescence including the TritonX-100 lysis response. The results are shown in Table 2. TABLE 2Transfected Buffer LPA MCH MCH cDNA (ECB) (1 μM) (1 μM) (1 μM) No Aeg 00 0 0 AEQ 2.7 5.24 1.9 3.43 AEQ + 2.76 6.83 26.4 26.6 MCHR 1

[0083] Transfection of the reporter gene aequorin into GOTO cellspermitted the detection of functional MCHR1 when co-transfected with acDNA encoding the human MCHR1 using 1 μM MCH to evoke a bioluminescentresponse. This observation indicates that neuroblastoma cells expressingMCHR1 are appropriate host cells for expressing the MCHR1 gene. In theabsence of exogenous MCHR1, no signal over background (buffer injectiononly, ECB) could be observed suggesting that the level of MCHR1naturally present in GOTO cells is insufficient to permit detectionusing the employed conditions. A control response evoked by theapplication of 1 μM lysophosphatic acid (LPA) suggests the presence ofan edg receptor (Im, et al., 2000. J. Biol. Chem. 275, 14281-14286), onGOTO cells linked to calcium mobilization.

[0084] Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1 9 1 353 PRT Human 1 Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly ProAsn Ala Ser Asn 1 5 10 15 Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser AlaGly Ser Pro Pro Arg 20 25 30 Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile MetPro Ser Val Phe Gly 35 40 45 Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn SerThr Val Ile Phe Ala 50 55 60 Val Val Lys Lys Ser Lys Leu His Trp Cys AsnAsn Val Pro Asp Ile 65 70 75 80 Phe Ile Ile Asn Leu Ser Val Val Asp LeuLeu Phe Leu Leu Gly Met 85 90 95 Pro Phe Met Ile His Gln Leu Met Gly AsnGly Val Trp His Phe Gly 100 105 110 Glu Thr Met Cys Thr Leu Ile Thr AlaMet Asp Ala Asn Ser Gln Phe 115 120 125 Thr Ser Thr Tyr Ile Leu Thr AlaMet Ala Ile Asp Arg Tyr Leu Ala 130 135 140 Thr Val His Pro Ile Ser SerThr Lys Phe Arg Lys Pro Ser Val Ala 145 150 155 160 Thr Leu Val Ile CysLeu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr 165 170 175 Pro Val Trp LeuTyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val 180 185 190 Gly Cys GlyIle Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe 195 200 205 Thr LeuTyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile 210 215 220 ThrAla Ala Tyr Val Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala 225 230 235240 Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg 245250 255 Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr260 265 270 Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr LeuThr 275 280 285 Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr AlaAsn Ser 290 295 300 Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu ThrPhe Arg Lys 305 310 315 320 Arg Leu Val Leu Ser Val Lys Pro Ala Ala GlnGly Gln Leu Arg Ala 325 330 335 Val Ser Asn Ala Gln Thr Ala Asp Glu GluArg Thr Glu Ser Lys Gly 340 345 350 Thr 2 1062 DNA Human 2 atggacctggaagcctcgct gctgcccact ggtcccaacg ccagcaacac ctctgatggc 60 cccgataacctcacttcggc aggatcacct cctcgcacgg ggagcatctc ctacatcaac 120 atcatcatgccttcggtgtt cggcaccatc tgcctcctgg gcatcatcgg gaactccacg 180 gtcatcttcgcggtcgtgaa gaagtccaag ctgcactggt gcaacaacgt ccccgacatc 240 ttcatcatcaacctctcggt agtagatctc ctctttctcc tgggcatgcc cttcatgatc 300 caccagctcatgggcaatgg ggtgtggcac tttggggaga ccatgtgcac cctcatcacg 360 gccatggatgccaatagtca gttcaccagc acctacatcc tgaccgccat ggccattgac 420 cgctacctggccactgtcca ccccatctct tccacgaagt tccggaagcc ctctgtggcc 480 accctggtgatctgcctcct gtgggccctc tccttcatca gcatcacccc tgtgtggctg 540 tatgccagactcatcccctt cccaggaggt gcagtgggct gcggcatacg cctgcccaac 600 ccagacactgacctctactg gttcaccctg taccagtttt tcctggcctt tgccctgcct 660 tttgtggtcatcacagccgc atacgtgagg atcctgcagc gcatgacgtc ctcagtggcc 720 cccgcctcccagcgcagcat ccggctgcgg acaaagaggg tgacccgcac agccatcgcc 780 atctgtctggtcttctttgt gtgctgggca ccctactatg tgctacagct gacccagttg 840 tccatcagccgcccgaccct cacctttgtc tacttataca atgcggccat cagcttgggc 900 tatgccaacagctgcctcaa cccctttgtg tacatcgtgc tctgtgagac gttccgcaaa 960 cgcttggtcctgtcggtgaa gcctgcagcc caggggcagc ttcgcgctgt cagcaacgct 1020 cagacggctgacgaggagag gacagaaagc aaaggcacct ga 1062 3 8483 DNA Human CDS(4208)...(4289) CDS (5504)...(6482) 3 atctctgcta aaattagccg ggcatggtggcacatgcttg taatcccagc tactctggag 60 gctgaagcca gagaatcact tgaaccaggaggcagaggtt tcagtgagct gagatcatac 120 cactgcactc cagcctgggc gacagagcaagactctgtct caaatgaaaa aatacataca 180 taaattaatt aattaaaaaa gaaaggagacagtttattga caggggagaa ctctcctgca 240 acacaggatg tagctggagc cagtcctaacatgcttctgg gatagctcca tgaaatggtt 300 tacaataata aggtggggat gctgggatggccattgcaga gttttgggat tttttgtgca 360 tagtaaaata cacataaaat ttgcctcttcactttatata ttgtggtagg atatacgtaa 420 cataaaattt accattttaa ccacttttaagtgtccaatt tagtggcatt aagtacgttc 480 acaatgttgt gcagccatta tcactgtccatttctagaac tttttttttt ttaagatgga 540 ttttcgctat tgttgcccag gctggagggtaatggcacaa tctcagctca ctgcaaccct 600 cacctcccgg gttcaagcaa ttctcctgcctcagcctcct gagtagctgg gactacaggc 660 acatgccacc atgcccggct aatttttgtatgcttagtag aggcagggtt tcaccatgcc 720 agccaggctg gtttcaagct cctgacctcaggtgatccgc ctgccttggc ctcccaaagt 780 gctgggatta gaggcaagag tcaccacacctggccagcta cacactttta aacaacaaga 840 tctcatgagc actcactcat tatcacaaaaacaggaaggg ggaaatctgt tcccatggtc 900 caatcacctc ccaccaaaca tgcttcctcacttccttaag gacttttctg gaagtgcttc 960 tcagtgaggc cttcctctac tgcaaaaagcttcccctgcc acaaagagct tctttttttt 1020 tgagacagag tctcgttctg tcacccaggctggagtgcag tggcgtgatc tcagctcact 1080 gcaatctcca cctcctgggt tcaagcgattctcatgcttc agcctcccaa gtagctggga 1140 ttacaggtgc ttgccaccac acctggctaatttttgtaca tttagtagac acagggtttc 1200 accatgttgg ccaggctggt ctcaaactcctgacctcagt caatccaccc gcctcagcct 1260 cccaaaatgt tgggattaca ggcgtgagccactgtgcccg gccaagactg atcctttcaa 1320 aacatgtctg atcatgtctt ttctcagctgaaaaccccct ggtgactccc atttccccta 1380 aaggcaaagc caaagtgctt atactggctggcaaggcctg gcagaatctg cccttcctgc 1440 tccttcctct ctgatgacag ctcctgctaatctctactat agccacagtg ctctccagag 1500 tcacactgca cactcctgcc acagggcctctgcactgaca atttccatgg cagggaatgc 1560 tggtcctcca gaatatttct tcttcttttttttttttttg agacggcgtc tcactctgtc 1620 acccaggctg gagtgcaatg gcatgatctcggctcactgc aatttccacc tcccgggttc 1680 aagcgattct cctgcctcag cctcccaagtagctgggact acaggcgccc gccaccacgc 1740 ccagctaatt tttatatttt taatagagagggggtttcac cttgttggcc aggattgtct 1800 cgatctcttg acatcatgat ccgcctgcctcggcctccca aagtgctggg atttcaggcg 1860 tgagccactg cgcccggcca ctccagaatatttctaagca tgcttcctca cttccttcag 1920 gacttttctc agagaggcct tccttgatgatcaatcctct ataaaatggt aacctccacc 1980 tctcttggta ttcctgggcc ctctcaccctgctttaagtt ttttacagca attgccaata 2040 tcataagtta tctttatgtg gagtatatattttttctttt ttttgagacg tagtctcgct 2100 ctgtcaccca ggctggagtg cagtggcgtggtctcggctc actgcaagct ccgcctcccg 2160 ggttcacgcc attctcctgc ctcagcctcccgagtagctg ggactacagg cacccgccac 2220 cacgcccggt taattttttg tattttttaatagagatggg gtttcaccat gttagccagg 2280 atggtctcga tctcctgacc tcgtgatctgccagcctcgg cctcccaaag tgctggtatt 2340 acaggtgtga gccactgcgc ccggccatattttttctttt ttctgagaca gggtctttct 2400 gtgtcgccca ggctggagtg cagtggcccaatctcagctc acttcaatct ctgcctcctg 2460 gattcaggcg attctcctgc ttcagctgcttaagtagctg gaattacagg cagctgccaa 2520 catgtccggc taatttttgt atttttgtagagacggggtt tcaccatgtt gtccaggctg 2580 gtcttgaact cctgagctca agcaatccacccaccttggc ttcgcaaagt gctgggatca 2640 caggcatgag ccactgcacc tggcccaaccctgttcttca gaatcaccct gcacacttcc 2700 tgccgcacgg cctttgcact ggcgatttccacagccagga atgctggtcc tccagaatat 2760 ttctaagcat ggttcctcac ttccttcaggacttttctgg aagcgcttct cagtgaggtc 2820 ttcctcgatg atcaatccta tataaaatagcaacctccac ctctgctggt attcaggggc 2880 cagctcaccc tgctttaagt ttttcatagtaattgccaat accatgaatt gtcttttgag 2940 gggttttctg tctccccgca acttgaatgcaatcttcatg aggacaggga ctttatcccc 3000 caccccaccc ccactttttt ttcttttttttttactgcaa aacctagaac agtgcttggc 3060 aggtagtaag tactcaatga atgtttgttggatgaaccca ataagtaaac aagatagagg 3120 acacatgtag ggatctgccc gtgaacctcgacccctggct cctgagtctg gcagtgggtg 3180 cagtggcagc tcctgtctgt gagggccccagaggctggca gcaggatgcc tccgtggaaa 3240 attcccttaa cgcctttgcc tctgcagctgttcctccggg atgatctctt tgggggatca 3300 tgctcagata tttgtctcaa agagtcccaggccaaacctc agggacctca gagcgtttag 3360 aaaaataaca cctctgtgag cttggtccaggcagatccca tgcagagagg agtttgtccc 3420 cttccagtcc ccgaggtcct ggctattgccagcatggagt gacctgtgtc acctctgagt 3480 gccaggcaag ggttcagcag ctgacgactcagcttctgca ggatgctggc agcatagcca 3540 gcgagatagt tggaagccgt cagggcacagggaaggggcc gagggtgccc tgagtgtgca 3600 tggggggcag ccctgctgca gtccaagcctttgattccca agctatgtgc acagtttcct 3660 ctggactctg ccatgtggcc cagccacccatacctggaat aggggctaag ccaagctgct 3720 ctctcctcca aagggaggca gcctgtgtgctttgtccgtt tgcctttgca gagacctcga 3780 tcttcacgca aggcaagcag cagcccctgtaagcacacga gacaatccca agtgtcagtg 3840 ggaaggagat ccctttcctg atggggctgcctgtgtccag tccctcccag cttccccagg 3900 gccctggggc tctgcaggca ttcagaagtggaagccagcc acagcctggg actgaagagg 3960 ttaatgtgca tctgcctccg aatgttaatgtgtctaggtg atgtcagtgg gagccatgaa 4020 gaagggagtg gggagggcag ttgggcttggaggcggcagc ggctgccagg ctacggagga 4080 agaccccctt cccaactgcg gggcttgcgctccgggacaa ggtggcaggc gctggaggct 4140 gccgcagcct gcgtgggtgg aggggagctcagctcggttg tgggagcagg cgaccggcac 4200 tggctgg atg gac ctg gaa gcc tcgctg ctg ccc act ggt ccc aac gcc 4249 Met Asp Leu Glu Ala Ser Leu Leu ProThr Gly Pro Asn Ala 1 5 10 agc aac acc tct gat ggc ccc gat aac ctc acttcg gca g gtgagttgac 4299 Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu ThrSer Ala 15 20 25 tgggagccct ccctcctctg ggctgtgggt ggaaaatggg aaggtttcacccctgagcca 4359 aactgcttgg gaaactttat cacagttctt ggggacaaga tctgtggtctgctttgctct 4419 gaggggcagg agaaaagggg gcaatggtcc gcaggggcag acgggcaggagcagagcagg 4479 gggcgaaggc atattcagaa tggcaaggaa ggggggccag ccgtgagacagcaggggaag 4539 gctcgctgct gggttccaaa gatgcttggc agaaaaaatt ccaggctggaaaagcaagcg 4599 agagaagctg gagggtggta tgtgggagac agctgggggc tcactcctgcactgttagcc 4659 tcagcttttt actcccactt ggatgatgag gtctgagaca tccttactgccacctgggag 4719 aggccctggg aagggaagac ttcacagagc catgagggga ttaacttttctggtgaatta 4779 agcttcctga catttccaga gctgcggtgc cctgggattc cagctttgaaggagaaagga 4839 aggaaggaaa agaggaaagg cttatgtaga taatttttcc aggctgctgagctccaacag 4899 acagtttctg tctctgcttc actcaagaag cccaggctca gaagataccaatcaaggaaa 4959 tccccgctag gaagcctggg gtagggagag ctgctggctt gaccagggcacagccggcaa 5019 aagcctctac aagacagtca cccacagata tgcccaagaa tcagtacacagtttccaacc 5079 agagatctcc aaaatgaaac actcagggct acacatagga aaagcacgcacacacacaca 5139 cacacacata cacagacact tacttttgtg tccttctggc tatgctgacgagttttcctg 5199 gtgaagcccg gggctcacag agtaatctct gcagacaact gtggttcttgcctctggtgc 5259 ctgcaggagg caggcatgtt gtgtccttcc aagacagatg gctcagggcactctggtagg 5319 attcaccagg aaactcatgg agaagggaaa agggacaaga ttagcaacagtgaagggagg 5379 gagaatggtg ggagaggatt ccagatgaac ggtgggtcgc tggaggctgagcatgccagc 5439 aggatgtcag ttctcagagc aaagcccatg tcaaacagcc aacgcttgctccttctgtcc 5499 ccag ga tca cct cct cgc acg ggg agc atc tcc tac atc aacatc atc 5547 Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile30 35 40 atg cct tcg gtg ttc ggc acc atc tgc ctc ctg ggc atc atc ggg aac5595 Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn 4550 55 tcc acg gtc atc ttc gcg gtc gtg aag aag tcc aag ctg cac tgg tgc5643 Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys 6065 70 aac aac gtc ccc gac atc ttc atc atc aac ctc tcg gta gta gat ctc5691 Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu 7580 85 90 ctc ttt ctc ctg ggc atg ccc ttc atg atc cac cag ctc atg ggc aat5739 Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn 95100 105 ggg gtg tgg cac ttt ggg gag acc atg tgc acc ctc atc acg gcc atg5787 Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met 110115 120 gat gcc aat agt cag ttc acc agc acc tac atc ctg acc gcc atg gcc5835 Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala 125130 135 att gac cgc tac ctg gcc act gtc cac ccc atc tct tcc acg aag ttc5883 Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe 140145 150 cgg aag ccc tct gtg gcc acc ctg gtg atc tgc ctc ctg tgg gcc ctc5931 Arg Lys Pro Ser Val Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu 155160 165 170 tcc ttc atc agc atc acc cct gtg tgg ctg tat gcc aga ctc atcccc 5979 Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro175 180 185 ttc cca gga ggt gca gtg ggc tgc ggc ata cgc ctg ccc aac ccagac 6027 Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp190 195 200 act gac ctc tac tgg ttc acc ctg tac cag ttt ttc ctg gcc tttgcc 6075 Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala205 210 215 ctg cct ttt gtg gtc atc aca gcc gca tac gtg agg atc ctg cagcgc 6123 Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg220 225 230 atg acg tcc tca gtg gcc ccc gcc tcc cag cgc agc atc cgg ctgcgg 6171 Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg235 240 245 250 aca aag agg gtg acc cgc aca gcc atc gcc atc tgt ctg gtcttc ttt 6219 Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val PhePhe 255 260 265 gtg tgc tgg gca ccc tac tat gtg cta cag ctg acc cag ttgtcc atc 6267 Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu SerIle 270 275 280 agc cgc ccg acc ctc acc ttt gtc tac tta tac aat gcg gccatc agc 6315 Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala IleSer 285 290 295 ttg ggc tat gcc aac agc tgc ctc aac ccc ttt gtg tac atcgtg ctc 6363 Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile ValLeu 300 305 310 tgt gag acg ttc cgc aaa cgc ttg gtc ctg tcg gtg aag cctgca gcc 6411 Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro AlaAla 315 320 325 330 cag ggg cag ctt cgc gct gtc agc aac gct cag acg gctgac gag gag 6459 Gln Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala AspGlu Glu 335 340 345 agg aca gaa agc aaa ggc acc tg atacttcccc tgccaccctgcacacctcca 6512 Arg Thr Glu Ser Lys Gly Thr 350 agtcagggca ccacaacacgccaccgggag agatgctgag aaaaacccaa gaccgctcgg 6572 gaaatgcagg aaggccgggttgtgaggggt tgttgcaatg aaataaatac attccatggg 6632 gctcacacgt tgctggggaggcctggagtc aggtttgggg ttttcagata tcagaaatcc 6692 ccttggggga gcaggatgagacctttggat agaacagaag ctgagcaaga gaacatgttg 6752 gtttggataa ccggttgcactatatctgtg agctctcaaa tgtcttcttc ccaaggcaag 6812 aggtggaagg gtactgactgggtttgttta aagtcaggca gggctggagt gagcagccag 6872 ggccatgttg cacaaggcctgagagacggg aaagggcccg atcgctcttt cccgcctctc 6932 actggtgcga tggaaggtggcctttctccc aagctggtgg ataatgaaaa ataaagcatc 6992 ccatctctcg gcgttccagcatcctgtcaa tttccctttt gctctagagg atgcatgttt 7052 atttgagggg atgtggcactgagcccacag gagtaaaagc ccagtttgct aggaggtctg 7112 cttactgaaa acaaggagacctggggtggg tgtggttggg ggtcttaaaa ctaataaaag 7172 ctggggtcgg ggggcttttgcagctctggt gacattctct ccacggggca catttgctca 7232 gtcactaatc cagcttgagtgtccgtgtgt tctgcatgtg caggggtcat tctagtgccc 7292 ggtgtgttgg catcatctttttgctctagc ccttcctctc caaaataaaa tcaaataaag 7352 gaaaatctcc acccacatcactctggatgt tcttgtggac ttgggggtgg gtgtgggctg 7412 gggcggggaa ggtgggcagcagaaaagaga aagaggggtc acttggttgt ggaatttaga 7472 tcttggttca tgctgcatttttaggaagca tgaagcaggg atctgttttt ctgaacccac 7532 agggaggatt cagtggcataaatggaatta ctggggattc attagatttt gcagttctgc 7592 tgctgggctt gttcttgggactcagcttcc tgtcttctgc acaaaatccc ctgggctttg 7652 ttgtcatcag tgatgagtcctcaggcccca agtcccaacc cccactcccc cgcctcaacc 7712 ctcacccccg ctgagtcaccagccgcagag ccagctctta gggcagctga agcctctctg 7772 ctttctacag ctgagatctggtgtgggcac cttgaacaga agattacagc cggggccact 7832 gggaggcagc cacgactgctgcttggctgc tgcttcttgg tgtcttcact gagaggggac 7892 tgggagccgt cagtgcagtgctcagcagac cttactgaga gagcgggaga aagctgcaag 7952 catcctgaaa gcaggggcagcagcacagcc tgttcctctt cagagctgca gcagggagcc 8012 tcttcagaaa acttctgggcagcttccttg ggtcctgggg actttttttt tgagacagag 8072 tatcgctctg tcgcccaggctgaagtgcag tggtgtgatc tctgctcact gcaagctccg 8132 cctcccgggt tcacaccattctcctgcctc agcctcccga gtagctggga ctgcaggcat 8192 ccgccaccac gcccggctaatttttgtatt tttagtaaag acggggtttc accatgttag 8252 ccaggatggt ctcgatctcctaacctcatg atctgcctgc ctcggcctcc caaggtgctg 8312 agactgcagg cgtaagccaccgtgcccggc tttttttttt tttttttttt ttggacacag 8372 ggccttgccc tgttgcccaggctgtagtgc aggtgacatg atcacagctc actgcagcct 8432 cgacctccca gattcaagcaatcctcccac ctcagcctcc caagtagctg g 8483 4 24 DNA Artificial SequenceOligonucleotide Primer 4 atggacctgg aagcctcgct gctg 24 5 21 DNAArtificial Sequence Oligonucleotide Primer 5 gccagcaaca cctctgatgg c 216 23 DNA Artificial Sequence Oligonucleotide Primer 6 ggccccgataacctcacttc ggc 23 7 22 DNA Artificial Sequence Oligonucleotide Primer 7gaggagatct actaccgaga gg 22 8 22 DNA Artificial Sequence OligonucleotidePrimer 8 gcccatgagc tggtggatca tg 22 9 24 DNA Artificial SequenceOligonucleotide Primer 9 gtggacagtg gccaggtagc ggtc 24

What is claimed is:
 1. A neuroblastoma cell or a skin cell carcinomacomprising a recombinant melanin-concentrating hormone receptor 1(MCHR1) gene that expresses functional MCHR1.
 2. The cell of claim 1,wherein said cell is a human neuroblastoma cell.
 3. The neuroblastomacell of claim 2, wherein said recombinant MCHR1 gene is present in theneuroblastoma cell genome and comprises endogenous nucleic acid encodingfor MCHR1 transcriptionally coupled to an exogenous promoter.
 4. Theneuroblastoma cell of claim 3, wherein said exogenous promoter is a CMVpromoter.
 5. The neuroblastoma of claim 3, wherein said cell furthercomprises a recombinant gene encoding for aequorin.
 6. The neuroblastomacell of claim 3, wherein said neuroblastoma cell is selected from thegroup consisting of: GOTO, CHP-212, CHP-243, SK-N-BE(2), and SH-SY5Y. 7.The cell of claim 1, wherein said cell is SCC-25.
 8. A neuroblastomacell or skin cell carcinoma having increased MCHR1 expression producedby a process comprising the step of coupling endogenous nucleic acidencoding for MCHR1 to an exogenous promoter.
 9. The cell of claim 8,wherein said promoter is a CMV promoter.
 10. The cell of claim 8,wherein said cell is a neuroblastoma cell selected from the groupconsisting of: GOTO, CHP-212, SK-N-BE(2), CHP-243, and SH-SY5Y.
 11. Thecell of claim 8, wherein said cell is a skin cell carcinoma.
 12. Thecell of claim 11, wherein said cell is SCC-25.
 13. A method of measuringthe ability of a compound to effect MCHR1 activity comprising the stepsof: a) providing said compound to the cell of any one of claims 1-12;and b) measuring MCHR1 activity.
 14. The method of claim 13, whereinsaid step (a) further comprises the presence of an MCHR1 agonist. 15.The method of claim 14, wherein said MCHR1 agonist is humanmelanin-concentrating hormone.
 16. The method of claim 13, wherein saidcell is a human neuroblastoma cell.
 17. The method of claim 16, whereinsaid recombinant MCHR1 gene is present in the neuroblastoma cell genomeand comprises endogenous nucleic acid encoding for MCHR1transcriptionally coupled to an exogenous promoter.